Chimeric rat produced using rat embryonic stem cells in the presence of an ES cell differentiation suppressant

#This patent application is the U.S. national phase of International Patent Application No. PCT/JP2010/071412, filed Nov. 30, 2010, which claims the benefit of Japanese Patent Application No. 2009-274008, filed Dec. 1, 2009, and Japanese Patent Application No. 2010-166571, filed on Jul. 23, 2010, which are incorporated by reference in their entireties herein.

#The present invention relates to a method of preparing a chimeric embryo with improved germline transmission efficiency, which uses a rat pluripotent stem cell, particularly a rat embryonic stem cell (hereinafter to be referred to as “ES cell”), a method of preparing a chimeric rat by using the chimeric embryo, a chimeric rat prepared by the method, and a medium useful for preparing a chimeric rat.

#ES cell is a nearly totipotent cell line and highly useful for the preparation of a genetically modified animal and the like. For example, a chimeric animal can be produced by injecting ES cells, in which a particular gene has been destructed, into normal host blastocysts to be mixed with the cells of the host embryos and returning the mixture to a uterus, and a genetically modified animal, in which the particular gene has been destructed (knockout animal), can be produced by crossing the obtained chimeric animals or offsprings and selecting the animals born.

#FIG. 1 shows photographs of outgrowths of blastocysts in an ES cell culture medium (a) without addition of the YPAC factors, and (b) with addition of the YPAC factors. Blastocysts at 4.5 days were seeded onto division-inactivated MEF. The zona pellucida was removed using the Tyrode buffer solution. (c) is a graphic representation showing the results of quantitative PCR analyses of Venus, Oct4, Nanog, Sox2, and Rex1 in inner cell mass (ICM). Seven days after seeding, RNA was extracted from the dome-like portion in the ICM induced from 7 or 4 blastocysts without addition of the YPAC factors and with addition of the YPAC factors. The data are means for three independent experiments, being relative gene expression levels in REF, ICM, and ICM+YPAC factors.

#FIG. 2-1 A drawing showing the results of an examination of the characteristics of rat ES cells. (a) shows photographs of colonies showing influences of Y-27632. Separated single cells (1×105 TgWW1 cells, number of passages 6) were seeded to a 6-well plate. The left panel shows colonies obtained with addition of MEF+YPAC factors to the culture medium, the central panel shows colonies obtained with addition of PAC factor, and the right panel shows colonies obtained with addition of Y factor. (b) shows photographs of alkaline phosphatase (ALP) staining of the same various cells. (c) is a photograph of Giemsa staining of 50 cells of TgWL2 (number of passages: 7). TgWL2 (number of passages: 7) exhibited the karyotype of 42 chromosomes including the XX sex chromosome. (d) is a graphic representation comparing gene expression patterns by scatter plot analysis. TgWW1 and REF are shown in the left panel, and TgWW1 and LL are shown in the right panel. An Agilent gene chip (whole rat genome microarray kit) was used for microarray analysis. The center line represents the equivalence curve; the lines above and below it show that the sample gene expression levels differed 2 fold. (e) is a graphic representation comparing the gene expression patterns for REF, TgWL1, TgWW1, and LL. These are the results of Q-PCR of Venus, Oct4, Nanog, Sox2, and Rex1 in rat ES cell lines. The data are means for three independent experiments, being relative gene expression levels in REF, TgWL1, TgWW1, and LL. (f) is a photograph showing a teratoma formation from rat cells. This is a teratoma generated by subcutaneously transplanting 2.6×106 TgWW1 (number of passages: 5) cells to an immunodeficient mouse.

#FIG. 2-2 (g) is a drawing showing the results of microarray analysis and hierarchical cluster analysis. A one-color microarray based gene expression analysis containing 41,000 genes (Agilent Technology) was used. The numerical values on the panel indicate correlation coefficients. (h) shows immunostaining charts of Oct4, Nanog, and Sox2 in rat ES cells (lower panels). The upper panels show DAPI staining images. (i) shows cross-sectional views of the three germ layers of teratoma induced from the TgWW1 ES cell line.

#The present invention provides a method of efficiently preparing a germline-transmitted chimeric rat using a rat pluripotent stem cell without being subject to limitations to a particular strain of a rat pluripotent stem cell or a strain of a host embryo. Described below are a rat pluripotent stem cell according to the present invention, a method of preparing a chimeric embryo and a chimeric rat, a method of preparing a genetically modified rat, and a kit for preparing a chimeric rat.

#1. Rat Pluripotent Stem Cell

#In the present invention, “a pluripotent stem cell” refers to a cell that maintains undifferentiated state and pluripotency, represented by ES cells and induced pluripotent stem cells (iPS cells). The ES cell may be an ES cell resulting from nuclear reprogramming of a somatic cell but is preferably prepared from rat early embryo by the method described later. Besides ES cells, examples include embryonic germ cells (EG cells) derived from primordial germ cells, multipotent germline stem cells (mGS cells) isolated from the testis, and the like. Preferably, the rat pluripotent stem cell to be used in the present invention is rat ES cell or rat iPS cell, more preferably rat ES cell.